Immunocytochemical localization of acid phosphatase in rat liver

Localization of acid phosphatase (ACPase) in rat liver was investigated by immunocytochemical techniques. Rat liver was fixed by perfusion and cut into thick tissue slices, which were embedded in Epon or Lowicryl K4M. For light microscopy (LM), semithin Epon sections were stained for the enzyme ACPase by an indirect immunoenzyme technique. For electron microscopy (EM), ultra-thin Lowicryl K4M sections were stained by a protein A-gold technique. By means of LM, granular reaction deposits were observed in hepatocytes and sinus-lining cells. Stained granules were present in the juxtanuclear cytoplasm, but they did not correspond to a typical staining pattern for the Golgi complex. EM revealed that gold particles indicating ACPase antigens were present on lysosomes and on some vesicles locating in the trans Golgi region. Endosomelike vesicles were strongly positive for the labeling. Golgi cisterna were mostly negative, but weak signals were noted in dilated sacules. The plasma membranes on the sinusoidal and bile canalicular sides were labeled by a few gold particles. The results indicate that ACPase is present in endosomes and in a restricted area of plasma membrane, as well as in the lysosomal system.     

Determinants on tmRNA for initiating efficient and precise trans-translation: some mutations upstream of the tag-encoding sequence of Escherichia coli tmRNA shift the initiation point of trans-translation in vitro.

tmRNA facilitates a novel translation, trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence. The mechanism underlying resumption of translation at a definite position is not known. In the present study, the effects of mutations around the initiation point of the tag-encoding sequence of Escherichia coli tmRNA on the efficiency and the frame of tag translation were assessed by measuring the incorporations of several amino acids into in vitro poly (U)-dependent tag-peptide synthesis. One-nucleotide insertions within the tag-encoding region did not shift the frame of tag translation. Any 1-nt deletion within the span of -5 to -1, but not at -6, made the frame of tag translation heterologous. Positions at which a single base substitution caused a decrease of trans-translation efficiency were concentrated within the span of -4 to -2. In particular, an A-4 to C-4 mutation seriously damaged the trans-translation, although this mutant retained normal aminoacylation and ribosome-binding abilities. A possible stem and loop structure around this region was not required for transtranslation. It was concluded that the tag translation requires the primary sequence encompassing -6 to +11, in which the central 3 nt, A-4, G-3, and U-2, play an essential role. It was also found that several base substitutions within the span of -6 to -1 extensively shifted the tag-initiation point by -1.     

A simplified and rapid limiting dilution assay of T lymphocytes

We report here the development of an alternative limiting dilution assay (LDA) of T lymphocytes (T cells). Blood mononuclear cells were first stimulated for 60 hr with PHA and then cultured in microwells in the presence of recombinant interleukin-2 without feeder cells. After 4 days of culture, wells were scored for proliferation. Clonal expansion of T cells followed the single-hit model of the Poisson distribution. The progenitor frequency (f) in the mononuclear cells and E-rosette-positive cells from normal donors were 0.082 +/- 0.025 (n = 12) and 0.236 +/- 0.029 (n = 5), respectively, but this was markedly decreased in patients who underwent marrow-ablative chemotherapy and autografts with blood hematopoietic stem cells. This LDA system should be of value in routine use for the evaluation of T cell proliferative activities.     

Reduced uptake and enhanced release of cadmium in cadmium-resistant metallothionein null fibroblasts.

Metallothionein (MT) is known to play a predominant role in the protection of cells from cadmium (Cd) toxicity. To investigate other factors involved in Cd resistance, we established Cd-resistant cell lines from simian virus 40-transformed MT null fibroblasts. Cd-resistant MT null cells, Cd-rA7 and Cd-rB5, developed approximately 10-fold resistance to Cd compared to parental cells, but showed no cross-resistance to Zn, Cu, Hg, Ni, As, cisplatin or H2O2. Accumulation of Cd in the resistant cells was 13-18% of that of parental cells after treatment with Cd for 24 h. A short-term experiment revealed that the rate of Cd incorporation into the Cd-resistant cells was suppressed, and the rate of Cd release was enhanced in the resistant cells compared with that of parental cells. These results indicate that the altered transport of Cd, slow uptake and rapid release, may confer resistance to Cd on the Cd-resistant cells established from MT null fibroblasts.     

5'-Nucleotidase in rat liver lysosomal membranes is anchored via glycosyl-phosphatidylinositol

We have previously demonstrated that 5'-nucleotidase, known as a plasma membrane enzyme, is also distributed both in rat liver tritosomal membranes and contents (J. Biochem. 101, 1077-1085, 1987). When the lysosomal membranes isolated from rat livers were incubated with phosphatidylinositol-specific phospholipase C purified from B. thuringiensis, about 70% of 5'-nucleotidase activity was released from the membranes. Judging from the result by phase separation with Triton X-114, the enzyme solubilized by the phospholipase C digestion showed a hydrophilic nature such as that of the tritosomal contents. Immunoblot analysis showed that the molecular weight of 5'-nucleotidase released from the lysosomal membranes by the phospholipase C digestion was almost identical with that of the enzymes from the Tritosomal contents. The above results showed that the phosphatidylinositol-specific phospholipase C-like enzyme in the lysosomes may be responsible for the conversion of the lysosomal membrane-bound 5'-nucleotidase to the soluble form present in the lysosomal matrix.